Liver
The liver is the major metabolic control organ of the human body that comprises thousands of minute lobules (lobuli hepatis), the functional units of the organ. Liver tissue contains two major differentiated cell types: parenchymal cells (i.e., hepatocytes) and non-parenchymal cells. The complex functions of liver are exerted to a large extent by hepatocytes, whereas non-parenchymal cells such as Kupffer cells, Ito cells and liver sinusoidal endothelial cells (LSEC) play important roles in supporting and providing supplies to hepatocytes. Mochida et al. (1996) Biochem. Biophy. Res. Comm. 226:176-179.
The liver acts as a guardian interposed between the digestive tract and the rest of the body. A major hepatic function involves effective uptake, storage, metabolism and distribution to blood and bile large amounts of substances such as carbohydrates, lipids, amino acids, vitamins and trace elements. Another function of the liver is the detoxification of xenobiotic pollutants, drugs and endogenous metabolites, through both phase I (oxidation/reduction) and phase II (conjugation) mechanisms.
Because of its essential role to life, liver dysfunction and diseases are often debilitating and life threatening. A number of acute or chronic pathological conditions are associated with structural and/or functional abnormalities of the liver. These include, but are not limited to, liver failure, hepatitis (acute, chronic or alcohol), liver cirrhosis, toxic liver damage, medicamentary liver damage, hepatic encephalopathy, hepatic coma or hepatic necrosis.
Many chemical and biological agents, either therapeutic or purely harmful, can induce liver damages and thus are hepatotoxic. The susceptibility of the liver to damage by hepatotoxic agents may be related to its primary role in metabolism or is a consequence of hypersensitivity reactions. Up to 25% of cases of fulminant hepatic failure may be the result of adverse reactions to medical agents. Hepatotoxic compounds are also an important cause of chronic liver disease including fatty liver, hepatitis, cirrhosis and vascular and neoplastic lesions of the liver. (Sinclair et al., Textbook of Internal Medicine, 569-575 (1992) (editor, Kelley; Publisher, J. B. Lippincott Co.).
Hepatotoxic agents may induce liver damage by cytotoxicity to the liver directly or through the production of toxic metabolites (this category includes the hypersensitivity reaction which mimics a drug allergy); cholestasis, an arrest in the flow of bile due to obstruction of the bile ducts; and vascular lesions, such as in veno occlusive disease (VOD), where injury to the vascular endothelium results in hepatic vein thrombosis. Individual susceptibility to liver damage induced by hepatotoxic agents is influenced by genetic factors, age, sex, nutritional status, exposure to other drugs, and systemic diseases (Sinclair et al., Textbook of Internal Medicine, Supra).
In addition to normal growth during early development, liver tissue has a unique ability to regenerate at adult stage. Liver regeneration after the loss of hepatic tissue is a fundamental component of the recovery process in response to various forms of liver injury such as hepatotoxicity, viral infection, vascular injury and partial hepatectomy. Following partial hepatectomy, for example, the liver size is usually restored to its original mass within about six days. Liver growth and regeneration involves proliferation of both hepatocytes and non-parenchymal cells such as sinusoidal endothelial cells. Typically, hepatocytes are the first to proliferate, and other cells of the liver enter into DNA synthesis about 24 hours after the hepatocytes. Michalopoulos and DeFrances (1997) Science 276:60-65.
Factors for Liver Proliferation
Several growth factors and cytokines have been implicated as being able to induce liver regeneration, most notably hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor-α (TGF-α), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), basic and acidic fibroblast growth factors, CTGF, HB-EGF, and norepinephrine. Fujiwara et al. (1993) Hepatol. 18:1443-9; Baruch et al. (1995) J. Hepatol. 23:328-32; Ito et al. (1994) Biochem. Biophys. Res. Commun. 198:25-31; Suzuma et al. (2000) J. Biol. Chem. 275:40725-31; Michalopoulos and DeFrances (1997) supra. As one of the most potent liver mitogens, HGF was first identified as a factor capable of stimulating DNA synthesis in cultured hepatocytes but is now known to have multiple distinct functions on a variety of epithelial cells. Nakamura et al. (1984) Biochem. Biophys. Res. Comm. 122:1450; Russell et al. (1984) J. Cell. Physiol. 119:183-192. Scatter factor (SF), which enhances motility and invasiveness of certain cell types, was found to have identical amino acid sequence as HGF, leading to the designation HGF/SF. Stoker and Perryman (1985) J. Cell Sci. 77:209-223; Gherardi and Stoker (1990) Nature 346:228. HGF/SF is synthesized as an inactive, single-chain zymogen that is subsequently cleaved to produce an active, dimeric glycoprotein composed of a 69-kDa α-subunit and a 34-kDa β-subunit held together by a single disulfide bond. Nakamura et al. (1989) Nature 342:440-443; Roos et al. (1995) Am. J. Physiol. 268:G380-6.
All known biological effects of HGF are transduced via a single tyrosine kinase receptor, Met, the product of the Met protooncogene. HGF/SF acts predominantly on Met-expressing epithelial cells in an endocrine and/or paracrine fashion, to mediate such diverse biological activities as proliferation, branching, cell migration, morphogenesis and lumen formation. van der Voort et al. Adv. Cancer Res. 79:39-90 (2000). In the liver, HGF is expressed in non-hepatocyte cells such as Ito cells and LSECs, whereas met transcripts are strongly expressed in hepatocytes. Hu et al. Am. J. Pathol. 142:1823-1830 (1993). After chemical or mechanical liver injury, HGF levels sharply increase, leading to a strong hepatocyte proliferation. Horimoto et al. J. Hepatol. 23:174-183 (1995). Livers from transgenic mice with liver-specific overexpression of HGF are twice the size of livers of control animals and they regenerate much faster after partial hepatectomy. Sakata et al. (1996) Cell Growth Differ. 7:1513-1523; Shiota et al. (1994) Hepatol. 19:962-972. Furthermore, HGF null mutant mouse embryos fail to develop a fully functional liver, demonstrating the essential role of HGF during liver development. Schmidt et al. (1995) Nature 373:699-702. The continuous infusion of large doses (5 mg/kg/day) of HGF directly into the portal vein has been shown to result in a significant increase of relative liver mass in mice. Patijn et al. (1998) Hepatol. 28:707-16. While HGF was found to be a potent inducer of hepatocyte mitosis, however, it failed to induce proliferation of nonparenchymal cells including sinusoidal endothelial cells. Patijn et al., supra. In other biological contexts, conversely, HGF has been shown as a potent endothelial cell mitogen. Rosen and Goldberg (1997) In: Regulation of Angiogenesis. Rosen, E, Goldberg, ID, Eds. Springer Verlag. pp 193-208.
It has been suggested that substantially high HGF plasma concentrations may be required in order to promote liver growth in vivo (Roos et al. (1995) Am. J. Physiol. 268:G380-6). HGF, by virtue of its strong heparin-binding properties, is largely sequestered in extrahepatic tissues following intravenous administration (Zioncheck et al. (1994) Endocrinology 134:1879-87) and the co-administration of dextran sulfate is required for an effective liver-promoting action (Roos et al., 1995).
Angiogenesis and Liver
Angiogenesis is an important cellular event in which vascular endothelial cells proliferate, prune and reorganize to form new vessels from preexisting vascular network. There are compelling evidences that the development of a vascular supply is essential for normal and pathological proliferative processes (Folkman and Klagsbrun (1987) Science 235:442-447). Delivery of oxygen and nutrients, as well as the removal of catabolic products, represent rate-limiting steps in the majority of growth processes occurring in multicellular organisms. Thus, it has been generally assumed that the vascular compartment is necessary, albeit but not sufficient, not only for organ development and differentiation during embryogenesis, but also for wound healing and reproductive functions in the adult. However, recent evidence suggests that, at least in the mouse embryo, the vascular endothelium may have an inductive effect on liver (Matsumoto et al. (2001) Science 294:559-563) and pancreas organogenesis (Lammert et al. (2001) Science 294:564-567), even prior to the establishment of a blood flow. The mechanism of such induction is unknown.
Angiogenesis is also implicated in the pathogenesis of a variety of disorders, including but not limited to, proliferative retinopathies, age-related macular degeneration, tumors, rheumatoid arthritis (RA), and psoriasis. Folkman (1995) Nat Med 1:27-31. Regenerating liver, in analogy to rapidly growing tumors, must synthesize new stroma and blood vessels. Not surprisingly, therefore, many studies have focused on angiogenesis in liver development and regeneration, as well as the roles of many known angiogenic factors therein. Michalopoulos and DeFrances (1997) supra; Mochida et al. (1996).
Vascular endothelial cell growth factor (VEGF), a potent mitogen for vascular endothelial cells, has been reported as a key regulator of angiogenesis and vasculogenesis. Ferrara and Davis-Smyth (1997)Endocrine Rev. 18:4-25; Ferrara (1999) J. Mol. Med. 77:527-543. Compared to other growth factors that contribute to the processes of vascular formation, VEGF is unique in its high specificity for endothelial cells within the vascular system. Recent evidence indicates that VEGF is essential for embryonic vasculogenesis and angiogenesis. Carmeliet et al. (1996) Nature 380:435-439; Ferrara et al. (1996) Nature 380:439-442. Furthermore, VEGF is required for the cyclical blood vessel proliferation in the female reproductive tract and for bone growth and cartilage formation. Ferrara et al. (1998) Nature Med. 4:336-340; Gerber et al. (1999) Nature Med. 5:623-628.
In addition to being an angiogenic factor in angiogenesis and vasculogenesis, VEGF, as a pleiotropic growth factor, exhibits multiple biological effects in other physiological processes, such as endothelial cell survival, vessel permeability and vasodilation, monocyte chemotaxis and calcium influx. Ferrara and Davis-Smyth (1997), supra. Moreover, recent studies have reported mitogenic effects of VEGF on a few non-endothelial cell types, such as retinal pigment epithelial cells, pancreatic duct cells and Schwann cells. Guerrin et al. (1995) J. Cell Physiol. 164:385-394; Oberg-Welsh et al. (1997) Mol. Cell. Endocrinol. 126:125-132; Sondell et al. (1999) J. Neurosci. 19:5731-5740.
Substantial evidence also implicates VEGF's critical role in the development of conditions or diseases that involve pathological angiogenesis. The VEGF mRNA is overexpressed by the majority of human tumors examined (Berkman et al. J Clin Invest 91:153-159 (1993); Brown et al. Human Pathol. 26:86-91 (1995); Brown et al. Cancer Res. 53:4727-4735 (1993); Mattern et al. Brit. J. Cancer. 73:931-934 (1996); and Dvorak et al. Am J. Pathol. 146:1029-1039 (1995)). Also, the concentration of VEGF in eye fluids are highly correlated to the presence of active proliferation of blood vessels in patients with diabetic and other ischemia-related retinopathies (Aiello et al. N. Engl. J. Med. 331:1480-1487 (1994)). Furthermore, recent studies have demonstrated the localization of VEGF in choroidal neovascular membranes in patients affected by AMD (Lopez et al. Invest. Ophtalmo. Vis. Sci. 37:855-868 (1996)). Anti-VEGF neutralizing antibodies suppress the growth of a variety of human tumor cell lines in nude mice (Kim et al. Nature 362:841-844 (1993); Warren et al. J. Clin. Invest. 95:1789-1797 (1995); Borgström et al. Cancer Res. 56:4032-4039 (1996); and Melnyk et al. Cancer Res. 56:921-924 (1996)) and also inhibit intraocular angiogenesis in models of ischemic retinal disorders (Adamis et al. Arch. Ophthalmol. 114:66-71 (1996)). Therefore, anti-VEGF monoclonal antibodies or other inhibitors of VEGF action are promising candidates for the treatment of solid tumors and various intraocular neovascular disorders.
Human VEGF was obtained by first screening a cDNA library prepared from human cells, using bovine VEGF cDNA as a hybridization probe. Leung et al. (1989) Science, 246:1306. One cDNA identified thereby encodes a 165-amino acid protein having greater than 95% homology to bovine VEGF; this 165-amino acid protein is typically referred to as human VEGF (hVEGF) or VEGF165. The mitogenic activity of human VEGF was confirmed by expressing the human VEGF cDNA in mammalian host cells. Media conditioned by cells transfected with the human VEGF cDNA promoted the proliferation of capillary endothelial cells, whereas control cells did not. Leung et al. (1989) Science, supra.
Although a vascular endothelial cell growth factor could be isolated and purified from natural sources for subsequent therapeutic use, the relatively low concentrations of the protein in follicular cells and the high cost, both in terms of effort and expense, of recovering VEGF proved commercially unavailing. Accordingly, further efforts were undertaken to clone and express VEGF via recombinant DNA techniques. (See, e.g., Ferrara (1995) Laboratory Investigation 72:615-618 (1995), and the references cited therein).
VEGF is expressed in a variety of tissues as multiple homodimeric forms (121, 145, 165, 189, and 206 amino acids per monomer) resulting from alternative RNA splicing. VEGF121 is a soluble mitogen that does not bind heparin; the longer forms of VEGF bind heparin with progressively higher affinity. The heparin-binding forms of VEGF can be cleaved in the carboxy terminus by plasmin to release a diffusible form(s) of VEGF. Amino acid sequencing of the carboxy terminal peptide identified after plasmin cleavage is Arg110-Ala111. Amino terminal “core” protein, VEGF (1-110) isolated as a homodimer, binds neutralizing monoclonal antibodies (such as the antibodies referred to as 4.6.1 and 3.2E3.1.1) and soluble forms of VEGF receptors with similar affinity compared to the intact VEGF165 homodimer.
Several molecules structurally related to VEGF have also been identified recently, including placenta growth factor (PIGF), VEGF-B, VEGF-C, VEGF-D and VEGF-E. Ferrara and Davis-Smyth (1987) Endocr. Rev., supra; Ogawa et al. (1998) J. Biological Chem. 273:31273-31281; Meyer et al. (1999) EMBO J., 18:363-374. A receptor tyrosine kinase, Flt-4 (VEGFR-3), has been identified as the receptor for VEGF-C and VEGF-D. Joukov et al. (1996) EMBO. J. 15:1751; Lee et al. (1996) Proc. Natl. Acad. Sci. USA 93:1988-1992; Achen et al. (1998) Proc. Natl. Acad. Sci. USA 95:548-553. VEGF-C has recently been shown to be involved in the regulation of lymphatic angiogenesis. Jeltsch et al. (1997) Science 276:1423-1425.
Two VEGF receptors have been identified, Flt-1 (also called VEGFR-1) and KDR (also called VEGFR-2). Shibuya et al. (1990) Oncogene 8:519-527; de Vries et al. (1992) Science 255:989-991; Terman et al. (1992) Biochem. Biophys. Res. Commun. 187:1579-1586. Neuropilin-1 has been shown to be a selective VEGF receptor, able to bind the heparin-binding VEGF isoforms (Soker et al. (1998) Cell 92:735-45). Both Flt-I and KDR belong to the family of receptor tyrosine kinases (RTKs). The RTKs comprise a large family of transmembrane receptors with diverse biological activities. At present, at least nineteen (19) distinct RTK subfamilies have been identified. The receptor tyrosine kinase (RTK) family includes receptors that are crucial for the growth and differentiation of a variety of cell types (Yarden and Ullrich, Ann. Rev. Biochem. 57:433-478, 1988; Ullrich and Schlessinger, Cell 61:243-254, 1990). The intrinsic function of RTKs is activated upon ligand binding, which results in phosphorylation of the receptor and multiple cellular substrates, and subsequently in a variety of cellular responses (Ullrich & Schlessinger, 1990, Cell 61:203-212). Thus, receptor tyrosine kinase mediated signal transduction is initiated by extracellular interaction with a specific growth factor (ligand), typically followed by receptor dimerization, stimulation of the intrinsic protein tyrosine kinase activity and receptor trans-phosphorylation. Binding sites are thereby created for intracellular signal transduction molecules and lead to the formation of complexes with a spectrum of cytoplasmic signaling molecules that facilitate the appropriate cellular response. (e.g., cell division, differentiation, metabolic effects, changes in the extracellular microenvironment) see, Schlessinger and Ullrich, 1992, Neuron 9:1-20. Structurally, both Flt-1 and KDR have seven immunoglobulin-like domains in the extracellular domain, a single transmembrane region, and a consensus tyrosine kinase sequence which is interrupted by a kinase-insert domain. Matthews et al. (1991) Proc. Natl. Acad. Sci. USA 88:9026-9030; Terman et al. (1991) Oncogene 6:1677-1683.
There are compelling evidences suggesting that Flt-1 and KDR have different signal transduction properties and possibly mediate different functions. Moreover, the signals mediated through Flt-1 and KDR appear to be cell type specific. Recent studies have provided considerable experimental data indicating that KDR is the major mediator of the mitogenic, angiogenic and permeability-enhancing effects of VEGF (Ferrara (1999) Kidney Int. 56:794-814). VEGF stimulation leads to a robust auto-phosphorylation of KDR and activation of the MAPK cascade, which may directly contribute to endothelial cell proliferation (Kroll and Waltenberger (1997) J. Biol. Chem. 272:32521-7). In contrast, the function of VEGFR-1 has been less clear, and many apparently conflicting reports on its function exist in the literature. This molecule displays a very weak or undetectable tyrosine autophosphorylation in endothelial cells in response to VEGF (Gille et al. (2000) EMBO J. 19:4064-4073). Flt-1 has been shown to have inhibitory effects on endothelial mitogenesis in several biological contexts, including early embryonic development, either by acting as a “decoy” receptor that prevents VEGF binding to VEGFR-2 or by directly inhibiting VEGFR-2 activities. Park et al. (1994) J. Biol. Chem. 269:25646-54; U.S. Pat. No. 6,107,046 (Alitalo et al.); Fong et al. (1999) Development 126:3015-25; Zeng et al. (2001). J. Biol. Chem. 276:26969-79). Other studies suggest that VEGFR-1 may mediate recruitment of monocytes and endothelial cell progenitors to the tumor vasculature (Barleon et al. (1996) Blood 87:3336-43) (Lyden et al. (2001) Nat. Med. 7:1194-201). Thus, the importance of VEGFR-1 signaling in the vascular endothelium is largely unclear.
Recent studies have attempted to elucidate the molecular mechanisms of various physiological and pathological processes in the liver, particularly liver regeneration. There have been proposed two reciprocal paracrine communication systems existing in hepatic tissues between hepatocytes and non-parenchymal cells such as sinusoidal endothelial cells. In one direction, growth factors such as HGF/SF are released from non-parenchymal cells such as sinusoidal endothelial cells and Kupfer cells, bind to their receptors (such as the c-Met receptor) on hepatocytes, and in turn induce and promote hepatocyte proliferation. In the opposite direction, it is suggested that VEGF expressed in and secreted from hepatocytes acts as a stimulatory factor that binds to its receptors (KDR and Flt-1) on sinusoidal endothelial cells, thereby stimulating the proliferation and maintenance of the sinusoidal endothelial cells in the liver. Yamane et al. (1994) Oncogene 9:2683-2690 observed that the endogenous expression of VEGF and VEGF receptors (Flt and KDR) as well as HGF and c-Met are strictly regulated in a cell-type specific manner in liver: using a flt-1 cDNA as a probe, flt-1 mRNA was found to be expressed at very high levels in sinusoidal endothelial cells in normal rat liver, but was hardly detectable in hepatocytes. Similar expression pattern was found for KDR, although the expression level was much lower. Yamane et al. further observed that, in an in vitro cell culture system, VEGF demonstrated a remarkably specific growth-stimulatory activity as well as maintenance activity on the sinusoidal endothelial cells.
Mochida et al. (1996) Biochem. Biophy. Res. Comm. 226:176-179 conducted in vitro experiments to monitor the expression levels of VEGF and VEGFRs in isolated hepatic cells from normal livers or partially resected livers. They found that in 70% resected rat liver, expression of VEGF, Flt-1 and KDR were all significantly increased. And the timing of the expression peaks for Flt-1 and KDR suggested that the upregulation of VEGFRs may be involved in proliferation of sinusoidal endothelial cells during liver regeneration.
More recently, Ajioka et al. (1999) Hepatology 29:396-402 examined the fate of transplanted hepatic tissues in the presence of exogenous VEGF. Isolated hepatocytes of adult mice were transfected with VEGF gene in vitro then transplanted intraperitoneally (i.p.) in mice, into an area adjacent to the pancreas. The transplanted hepatocytes formed a large number of tissue aggregates in vivo. In vitro staining showed that these VEGF-transfected tissues underwent substantial proliferation and developed a significant vascular network therein. Thus, the results suggested that the expression of VEGF conferred the formation of a vascular network, which in turn may promoted tissue formation. The results, however, showed absence of any nonparenchymal cells or growth factors derived from them in the VEGF-transfected, transplanted hepatic tissues.
Assy et al. (1999) J. Hepatol. 30:911-915 studied the effect of VEGF as an angiogenic factor in liver regeneration following partial hepatectomy in rat. Rats undergoing 30% partial hepatectomy were administered intravenously (i.v.) VEGF and sacrificed at 24, 36 and 48 hour postoperatively. Whilst the study showed increased DNA synthesis activities of hepatocytes in the VEGF-treated rats at 36 and 48 h after PHx, and suggested that stimulation of neovascularization by VEGF is important during liver regeneration, no statistically significant changes in restituted liver mass were observed in VEGF-treated rats as compared to control rats without VEGF treatment.